Evidence for a direct action of IL-10-activated STAT3 in modulating TNF-a, CXCL8 and IL-1ra gene transcription in LPS-treated neutrophils by protein synthesis-independent and dependent mechanisms

The anti-inflammatory action of IL-10 primarily consists in limiting the production of proinflammatory cytokines and chemokines and promoting the release of anti-inflammatory molecules from phagocytes activated by agonists of Toll-like receptors. We have recently shown that the ability of IL-10 to rapidly exert its full array of anti-inflammatory effects on human neutrophils is dependent upon exposure of these cells to LPS for at least 3-4 hours. Here, we demonstrate that, in neutrophils “preconditioned” by LPS, IL-10 primarily targets the transcription of TNF-, CXCL8 and IL-1ra genes in a rapid and protein synthesis-independent manner, as revealed by Primary Transcript (PT) real-time RT-PCR. We also show that the ability of IL-10 to maintain repressed, but not to initiate, LPS-induced TNF- and CXCL8 transcription results impaired if neutrophils are exposed to the protein synthesis inhibitor cycloheximide (CHX), in coincidence with a reduced ability of IL-10 to induce STAT3 tyrosine phosphorylation. Importantly, inhibition of IL-10-induced STAT3 activation and IL-10-suppression of LPS-induced TNF- and CXCL8 mRNA expression by a prolonged exposure to CHX was observed to occur also in human monocytes and was caused by a defective IL-10-mediated activation of Jak1 and Tyk2 kinases, downstream of the IL-10R. Taken together, our findings demonstrate that protein synthesis inhibition reverts the suppressive effects of IL-10 on LPS-induced cytokine gene expression by interrupting the signaling pathway upstream of STAT3 activation. These data challenge the concept of the requirement of an IL-10-induced mediator to execute IL-10 anti-inflammatory program.